Capillary tube printing tips for microarray printing

ABSTRACT

A microarray contact printing is formed from at least one capillary tube. The tip has concentric reservoir and printing capillary tubes, with a first capillary tube ( 24 ) and a second capillary tube ( 22 ) having an inner bore ( 26 ) with an inner diameter larger than an outer diameter of the first capillar tube ( 24 ) so that the second capillary tube ( 22 ) partially overlaps a proximal end of the first capillary tube ( 24 ). The first capillary tube ( 24 ) has a contact surface ( 36 ) at a distal end. The inner bore of the first capillary tube ( 24 ) is adapted for drawing the printing solution retained in the second capillary tube ( 22 ) and depositing a drop of a solution on a printing substrate when the contact surface ( 36 ) is moved proximate the substrate.

A portion of the disclosure of this patent document contains materialthat is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and Trademarkoffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

TECHNICAL FIELD

The present invention relates generally to devices and methods used formicroarray printing. More particularly, this invention pertains toprinting tips used for depositing spots of liquid material across amicroarray printing substrate.

BACKGROUND ART

DNA microarrays and other massively parallel screening technologies areredefining the approach to discovery in biomedical research. One keyaspect to interpreting these parallel screening approaches is theuniformity of conditions across the probe screen. Significantvariability across DNA microarrays is often observed and as a resulttypical DNA microarray hybridization results often discard much of thedata. One of the reasons for this is inadequate control of the chipmanufacturing process. The development of technologies which increasethe efficacy of DNA chip printing are therefore highly desirable. Suchadvances will establish lower limits of detectability for currentlyexisting procedures, as well as extend the utility of microarrays as aplatform technology into novel applications.

Most microarrays are produced by depositing nanoliter or picoliterquantities of a probe DNA solution across the array substrate to form100 to 200 um diameter spots. Large microarrays may contain thousands ofunique spots deposited at densities exceeding 4000 spots/cm². Thequantity of probe DNA comprising each spot is determined by thedeposition volume and resulting spot morphology. Variations in volumeand morphology affect the probe density of the spot, which can influenceimportant analysis parameters including hybridization specificity,dynamic range, and relative hybridization intensity among spots.Controlled and consistent deposition of the probe DNA solution onto themicroarray substrate is an important factor in accurate microarrayanalysis. Inter-spot consistency is another important factor. Variationsin deposition can alter spot characteristics which affect comparisonsbetween different spots. Variations from spot-to-spot necessitates theuse of self-normalizing experimental designs such as performed in twocolor differential gene expression. If spot-to-spot variation could bereduced, more robust experimental designs such as single labelhybridization studies would be much more feasible and produce morereliable data which could be compared across experiments.

Ideally, the method of depositing the probe solution onto the arraywould provide for precise and consistent control of both spot volume andmorphology. Precise control allows the quantity of deposited probesolution to be optimized for the selected method of attachmentchemistry. Consistency insures that all probe spots on the array havesimilar probe densities, size, and therefore similar hybridizationcharacteristics. Additionally, ideal characteristics include negligibleevaporation from the deposition device so that multiple sampledepositions by the same device over a period of time will have the sameconcentration. Printing strategies also require that loading the devicewith probe DNA solutions minimizes sample evaporation from the storageplates. This may be an important factor for high density arrays andensures that uniform sample concentrations are maintained for subsequentprints. For non-disposable tips, cleaning must be rapid and thorough toprevent sample carryover from consecutive loads of different probesolutions.

Most microarrays are currently produced by some form of contactprinting. Contact printing is popular because it offers a good balanceof cost and performance. It requires only a tiny fraction of thestart-up costs associated with photo-lithographic in-situ synthesismethods and is versatile enough to print large arrays with thousands ofunique spots. Compared to photolithographic and ink jet printingmethods, contact printing is less technically complex. Its simple andinherently passive operational requirements make it popular with manysmall microarray printing facilities such as academic core labs.

Current methods of depositing printing solution include solid pins,quill pins, and ink jets. Quill pins have a narrow slit at their tipwhich acts as a fluid reservoir during the printing process. Each timethe pin contacts the substrate, it deposits solution from the slitreservoir which holds a sufficient volume to print multiple spots from asingle load of probe solution. The volume and morphology of the spotsdepends on the equilibrium state between the pin tip and substrate andis influenced by factors like surface tension of the printing solution,and hydrophobic characteristics of the substrate and pin. Quill pins aremore difficult to clean and are not as consistent as solid pins becausethe delicate design of the quill pin geometry makes them susceptible todeformation. Special humidity conditions are often employed to reduceevaporation from the pins during printing to maximize spotrepeatability. Their chief advantage is speed resulting from theirability to print multiple spots from a single sample loading.

Solid pins have a small flat tip that is dipped into printing solutionbefore each deposition. When removed from the printing solution, a smallbubble of solution remains on the hydrophilic tip of the pin and isdeposited as the pin contacts the substrate. The volume and morphologyof the spot depends on the tip size, surface tension of the printingsolution and hydrophobic character of the substrate. Solid pins are easyto clean and provide good spot-to-spot consistency because of simple,rugged design that facilitates both inter- and intra-pin uniformity.Their primary drawback is slow deposition speed because they must bereloaded before printing each spot. Evaporation from the sample platesduring lengthy print runs can also be a problem.

Ink jet printing methods use a pressure pulse to eject a small quantityof probe solution through a small nozzle onto the array. The volume andmorphology of deposition is relatively consistent and can be controlledby adjusting the characteristics of the ejection pulse. The rate ofdeposition is extremely fast because the ink jet is not required tocontact the substrate. Evaporation of printing solution from inkjets isminimal so spot consistency over multiple depositions is excellent.Their main drawback is difficulty in cleaning and reloading. Typical inkjets are not designed for multiple samples so each unique probe solutionwould require its own ink jet device; a serious drawback for largearrays.

Microfluidic analysis of fluid transfer at this scale appears to be arelatively poorly understood process. However, several key features arereadily apparent. Pin loading by “capillary action” is one of thesurprising features of the fluid-surface interface at this scale.Theoretical expressions describing this have been attributed toRayleigh. For a hollow, round tube, the capillary rise height (definedas the distance fluid will travel up into a tube after submerging oneend in a reservoir) is governed by fluid properties and the subsequentcontact angle between the fluid and the pin capillary inner surface.This distance can be quite large. For example, in a 25 micron innerdiameter tube, water will rise to a height of 58 cm. The time toequilibrium has also been investigated and has been shown to bedependent on the fluid-surface interface characteristics. For the same25 micron tube with a length of 1 cm, the characteristic rise time isapproximately 300 ms.

Non-dimensional groups are another useful tool for describing thedominate forces in this microfluidic environment. The Weber number, We,describes the ratio of inertial to surface tension forces, and is givenby DV²ρ/g_(c)σ where D is the characteristic length, g_(c) is adimensionless constant, ρ is the density, σ is the surface tension, andV is the velocity. The Froude number, Fr, describes the ratio ofinertial to gravitational forces, and is given by V²/gL where V is againcharacteristic velocity, L is characteristic length, and g isgravitational acceleration. The ratio of these ratios (Fr/We) is arelative measure of the surface forces to gravitational forces and isgiven by g_(c)/L² pg. For a characteristic dimension of 25 microns, andfluid properties of water, this ratio is approximately 500. Since thisvalue is much greater than 1, this lends support to the idea thatsurface forces dominate.

The key characteristic is minimum variation among spots printed with asingle tip and minimum variation among spots printed with different tipsof the same design. Several other secondary factors are important forcommercial success. One of these is the overall speed of the printingprocess. Besides the obvious cost of robot time, sample evaporation andchanges in slide surface chemistry also need to be minimized. Anotherfactor is sample waste. Residual sample after printing is typically notreturned to the sample well. As noted above for a 25 microncharacteristic dimension, the volume loaded is 300 nl. Neglecting anyevaporation loss, this is sufficient to print >400 slides. A means tocontrol the volume loaded would be highly desirable. This would greatlyreduce the sample discarded and the number of slides obtainable from asample plate.

What is needed, then, is a printing tip for transferring sub-nanoliterliquid samples to a high density array printed on a flat surface whichis suitable for high-speed robotic printing. These tips will findapplication in DNA arrays as well as any other liquid handling processin which picoliter to nanoliter quantities are rapidly and preciselytransferred from a liquid reservoir to a flat solid surface.

DISCLOSURE OF THE INVENTION

The present invention provides significant improvements in microarrayprinting tip technology through the use of capillary tube printing tips.Several embodiments of capillary tube printing tips are disclosedherein.

The invention provides a simple printing tip. Tip loading is achieved bysurface forces and capillary action. Pin delivery is achieved bytouching the tip to the surface of a microarray printing substrate. Oneimportant aspect of the design is the flatness of the tip. This can beachieved on a glass tip by using an optical fiber polisher. A singleload can be dispensed without reloading onto many consecutive substratesurfaces. In addition, drop-to-drop variation is minimal. Unlike quillpins, the volume transferred to the surface equilibrates very rapidlymaking the deposition characteristics time independent. The simplicityof the design also suggests that pin-to-pin variation will beinsignificant. The tip design can be easily modified to deliver dropsover a range of sizes and volumes. These tips may be produced at lowcost so that they may be discarded between samples. This will obviatethe need for a wash cycle and eliminate cross contamination issues.

Preferably, means are provided in the design of the tip to provideincreased surface forces to the liquid material near the distal end ofthe tip so that the liquid material is continuously drawn down the tipfor deposition.

In a first embodiment of the invention, a tip for depositing spots of aliquid material on a microarray printing substrate includes a capillarytube having a distal end, a proximal end, and an inner bore. The innerbore has a bore opening at the distal and proximal ends of the tube. Theinner bore has an axial length and inner diameter adapted to receive andretain by capillary force an effective deposition volume of the liquidmaterial. Further, the distal end of the capillary tube has an annularcontact surface around the distal bore opening. The inner bore has aminimum diameter at the contact surface which expands to a largerdiameter towards the proximal end. The contact surface and distal boreopening are adapted for drawing the liquid material from the inner boreand depositing a drop of the liquid material on the printing substratewhen the contact surface is moved proximate the substrate.

In a second embodiment, a contact printing tip is formed from concentricreservoir and printing capillary tubes, with the second capillary tubehaving an inner bore with an inner diameter that is larger than an outerdiameter of the first capillary tube so that the second capillary tubepartially overlaps a proximal end of the first capillary tube. The firstcapillary tube also has having an inner bore in fluid communication withthe inner bore of the second capillary tube. The first capillary tubefurther comprises a contact surface at a distal end with the contactsurface surrounding an opening from the inner bore of the firstcapillary tube. The inner bore of the second capillary tube is adaptedto receive and retain an amount of the liquid material and the innerbore of the first capillary tube is adapted for drawing the liquidmaterial retained in the inner bore of the second capillary tube bycapillary action and depositing a drop of the liquid material on theprinting substrate when the contact surface is moved proximate thesubstrate.

In a third embodiment, a tip for depositing spots of a liquid materialon a microarray printing substrate includes a capillary tube having adistal end, a proximal end, and an inner bore. The inner bore has a boreopening at the distal and proximal ends of the tube. The inner bore hasan axial length and inner diameter adapted to receive and retain bycapillary force an effective deposition volume of the liquid material.Further, the distal end of the capillary tube has an annular contactsurface around the distal bore opening. In this uniform inner boregeometry the change in surface forces to achieve a more hydrophilicregion near the contact surface of the tube uses coatings applied to theinner bore so that surface forces near the distal end of the tip aregreater than surface forces in the remainder of the inner bore. Thecontact surface and distal bore opening are adapted for drawing theliquid material from the inner bore and depositing a drop of the liquidmaterial on the printing substrate when the contact surface is movedproximate the substrate.

Accordingly, it is an object of the present invention to provide animproved microarray printing tip.

Other and further objects, features and advantages of the invention willbe readily apparent to those skilled in the art upon a reading of thefollowing disclosure when taken in conjunction with the accompanyingdrawings.

FIG. 1(a) is a schematic cross-sectional elevation drawing of anembodiment of a capillary tube printing tip wherein a smaller diameterfirst (printing) capillary tube is partially overlapped by the distalend of a larger diameter second (reservoir) capillary tube so as toprovide a larger diameter liquid material reservoir located above thesmaller diameter active printing tip.

FIG. 1(b) is a schematic cross-sectional elevation drawing of theembodiment of a capillary tube printing tip of FIG. 1(a) mounted in atip holder and further showing the level of liquid material in the tipafter loading.

FIG. 1(c) is a schematic cross-sectional elevation drawing of a slightvariation of the embodiment of the capillary tube printing tip of FIG.1(a).

FIG. 2(a) is a side cutaway view of another embodiment of a microarrayprinting tip in accordance with the present invention, constructed froma glass capillary tube that tapers outward from the distal to theproximal end.

FIG. 2(b) is a photograph of the distal end of the microarray printingtip of FIG. 2(a) and further showing a deposited drop. The capillarytube shown in FIG. 2(b) is 90 microns in diameter. The drop produced byit is approximately 100 microns.

FIG. 3 is a side cutaway view of yet another embodiment of a microarrayprinting tip in accordance with the present invention, constructed froma glass capillary tube of uniform bore geometry but with a hydrophilictreatment applied to the inner bore surface to provide a gradient insurface forces from the distal to the proximal end.

BEST MODE FOR CARRYING OUT THE INVENTION

In FIGS. 2(a) and 2(b), a first embodiment of a microarray printing tipin accordance with the present invention is illustrated. The tip isconstructed as a capillary tube 10 having an inner bore 18. The innerbore 18 has an opening 19 at the distal (contact) end of the tube 10. Anannular contact surface 12 surrounds the opening 19. The inner bore 18has an inner diameter and an axial length that define a liquid reservoirvolume which, in cooperation with capillary and surface forces appliedat the interface between the liquid material and the inner surface ofthe inner bore, allows the tube 10 to receive and retain an appropriateamount of the liquid material.

As seen in FIG. 2(a), the diameter of the tube 10 and inner bore 18increases from the distal (contact or printing) end (FIG. 2(b)) to theproximal (reservoir) end to provide a desired gradient in surface forcesapplied to the liquid material. As will be understood by those skilledin the art, the capillary forces holding the liquid material within thetube 10 increase as the inner diameter of the tube decreases. Thevariation in inner bore diameter therefore functions as a means forproviding a desired surface force gradient, where surface forces are afunction of both the liquid/solid surface tension and capillary radius.When the contact surface 14 is moved proximate the surface of amicroarray printing substrate, the tube 10 draws and deposits a drop 16of the liquid material from the inner bore 18. The tube 10 shown in FIG.2 is a glass capillary tube having an outside diameter of 90 microns.However, other materials, such as stainless steel, can be used to formthe tube.

Also, depending on the characteristics of the liquid to be deposited, onthe desired geometry of the spot to be printed on the substrate, on theambient printing conditions including humidity, and on the nature of thesubstrate surface, the dimensions of the inner bore and contact surfacecan be varied. For example, for DNA microarray printing, the innerdiameter of the inner bore 18 can range from 10 to 2000 microns, with anaxial length of 100 microns to 10 cm. The diameter of the contactsurface (outer diameter of the tube at the distal end) can range from 10to 2000 microns.

FIG. 1(a) shows a second embodiment of a microarray printing tip 20constructed from concentric first and second capillary tubes 24 and 22.The second capillary tube 22 (reservoir tube) has an inner bore 26defining a liquid reservoir 34. The inner diameter of the inner bore 26is larger than the outer diameter of the first capillary tube 24 so thatthe second capillary tube 22 partially overlaps (at region 30) theproximal end of the first capillary tube 24.

The first capillary tube 24 (printing tube) has an inner bore 28 influid communication with the inner bore 26 of the second capillary tube22. The inner bore 28 has a bore opening 29 at the distal end of firsttube 24. An annular contact surface 36, preferably flat, is formed atthe distal end of the first capillary tube 24. The contact surface 36surrounds the opening 29 from the inner bore 28 of the first capillarytube 24.

The axial length and inner diameter of the inner bore 26 of the secondcapillary tube 22, in cooperation with capillary and surface forces, areadapted to receive and retain an amount of the liquid material withinthe reservoir 34. Similarly, the axial length, inner diameter, and innerbore surface of the first capillary tube 24 are adapted for drawing theliquid material retained in the reservoir 34 by capillary action anddepositing a drop of the liquid material on the printing substrate whenthe contact surface 36 is moved proximate the substrate. The largerinner bore diameter of the second capillary tube 22 as compared to theinner bore diameter of the first capillary tube 24 functions to providea surface force gradient that increases from the proximal to the distalend of the pin 20.

The first and second capillary tubes 26 and 24 can be made from glassor, in a preferred embodiment, from stainless steel. In the embodimentof FIG. 1(a), the second tube 22 has an outside diameter of 800 micronsand an inside diameter of 180 microns. The first capillary tube 24 hasan outside diameter of 170 microns so that it closely fits within theinner bore 26 of second capillary tube 22. The inner bore 28 of thefirst capillary tube has an inside diameter of approximately 100 μm.Again depending on the characteristics of the liquid to be deposited, onthe desired geometry of the spot to be printed on the substrate, on theambient printing conditions including humidity, and on the nature of thesubstrate surface, the dimensions of the inner bores and contact surfacecan be varied. For example, for DNA microarray printing, the innerdiameter of the inner bore 26 can range from 25 to 4000 microns, with anaxial length of 500 to 4000 microns. The inner diameter of the innerbore 28 can range from 5 to 250 microns, with an axial length of 500 to1500 microns. The diameter of the contact surface 36 (outer diameter ofthe tube) can range from 15 to 500 microns.

To assemble the tip 20, the first (printing) tube 24 is inserted adistance 30 into the second (reservoir) tube 22, and is held in placetherein by adhesive or the like as indicated at 32.

FIG. 1(b) shows the tip 20 fixed in a tip holder 35 after an effectivevolume of liquid material 37 has been loaded into the reservoir 34 andfirst inner bore 28 of the tip 20.

FIG. 1(c) illustrates a slightly different version of the embodiment ofthe microarray printing tip of FIG. 1(a) in which the outer diameter ofthe first capillary tube 24 is smaller than the inner diameter of thesecond capillary tube 22.

FIG. 3 shows a third embodiment of a capillary tube printing tip inaccordance with the present invention, constructed from a single glasscapillary tube 40 having an inner bore 42 of uniform geometry. Thedistal (contact) end of tube 40 has a contact surface 52 surrounding thebore opening 50. The inner bore 42 is sized and shaped to receive andretain by capillary force an effective deposition volume of the liquidmaterial. Similarly, the contact surface 52 and bore opening 50 areadapted for depositing a drop of the liquid material when the contactsurface 52 is moved proximate the printing substrate. A key designfeature of using two capillary tubes as shown in the embodiment of FIGS.1(a)-1(c) is the ability to modulate the relative strength of surfaceforces between the printing and reservoir capillary tubes. Thisfunctionality can also be attained by using a hydrophilic surfacetreatment to provide a gradient in surface forces along regions of asingle capillary tube. Thus, applying a surface treatment to the surfaceof the inner bore 42 at region 51 near the distal end of the tube 40would preferentially draw fluid from the less hydrophilic region 53 ofthe capillary tube 40, which would function as a reservoir. Modulatingthe relative strength of the surface forces along the axial length ofthe capillary tube 40 can then be used to control depositioncharacteristics. A number of commonly available silane compounds with arange of functional groups could be used to derivatize the interior ofthe capillary for this application. One example is N-octadecyl triethoxysilane.

With regard to each of the embodiments shown, it is important that thecontact surface be made as flat as possible. More specifically, anyvariation in flatness of the contact surface which would cause aseparation of the contact surface from the microarray substrate surfacewhich is being printed should be substantially less than the insidediameter of inner bore. Also, it is important that the contact surfacehave an appropriate surface finish so as to aid in wetting of thecontact surface. If the capillary tube is made from glass or stainlesssteel, a satisfactory contact surface can be provided through the use ofa high precision disc polisher of the type utilized to polish opticalfibers, using a 12 microgrit abrasive sheet.

In some applications, the concentric tube embodiment of FIG. 1 has beenfound to be preferable to the straight capillary tube of FIG. 3, due tothe interaction of the capillary forces in the smaller diameter innerbore 28 as compared to the larger diameter reservoir 34. As will beunderstood by those skilled in the art, the capillary forces holdingliquid within a tube increase as the inner diameter of the tubedecreases. In the embodiment of FIG. 1, a smaller stainless steelcapillary tube acts as the printing tip, drawing liquid from the largercapillary tube which acts as a reservoir. The smaller diameter of theprinting capillary tube exerts a greater surface force and automaticallydraws liquid solution from the reservoir capillary tube. Pin depositioncan be controlled by the diameter of the printing capillary tube (tocontrol spot diameter), and by the ratio of the printing and reservoircapillary tube radii (to control volume dispensed by pin). The reservoirvolume can be adjusted by changing the axial length of the reservoircapillary tube.

Thus, if a long, single diameter capillary tube is used, as liquid feedsout the contact (distal) end of the tube, the liquid remaining high inthe capillary tube will find it difficult to flow toward the contactend. On the other hand, when the majority of the volume of the liquid tobe transferred is placed in a larger diameter reservoir 34, then thesmaller diameter tube 24 can more easily draw fluid from the largerdiameter reservoir 34 due to the higher capillary forces acting on thesmaller diameter inner bore 26.

This simple design provides a number of manufacturing advantages.Critical geometry features of the capillary pin are automatically fixedby the constant diameters of the printing and reservoir capillarytubing. Controlling the diameter of the printing tip (to control spotsize) becomes trivial because grinding the tip flat does not affect tipdiameter. Manufacturing matched sets of capillary printing tips with thesame diameter and spot volume is easily accomplished by using the samegauge of tubing. Axial lengths of capillary and reservoir tubing appearto be less critical to printing characteristics. The technology formanufacturing capillary tubing stock well developed—high precisioncapillary tubing tolerances of plus or minus 5-6 um can be purchases ina range of suitable sizes. Diameters <150 um can be custom ordered.

Because critical dimensions of the capillary printing pins are fixed,the manufacturing precision required to produce the pins is greatlyreduced. One method of assembling capillary tube printing tips pins inaccordance with the present invention is using an adjustable alignmentjig. The jig is adjusted to hold the printing and reservoir capillarytubes in concentric alignment. The tubes are bonded together by wickinga small volume of 5-minute epoxy between the tubing overlap. After theadhesive cures, the assembly is removed from the jig and the printingcapillary tube is cut and ground to the desired length. A capillaryprinting tip can be assembled in approximately 20 minutes by thismethod, including 15 minutes for the adhesive to cure sufficiently.

Other methods can be used to improve the speed and precision ofmanufacturing. One approach is using an array of high precision jigs.This would allow many pins to be assembled simultaneously. Assemblyspeed could be increased by using photo-curing or heat curing adhesives.A variety of suitable adhesives are available which can be cured to aworking strength in a matter of seconds or minutes, including epoxyadhesives.

Custom manufactured reservoir tubing can be used with an inner diameterthat would fit the outer diameter of the printing capillary tube, and anouter diameter which would facilitate mounting to a printing head.Capillary tubing can be custom manufactured with extremely highprecision. The desired printing characteristics can be achieved andautomatically assembly facilitated if the concentric capillary tubeshave a concentric dimensional precision of less than 25 microns. Thiswould simplify manufacturing because achieving the required level ofaxial precision for the tubing assembly would become almost trivial.

The relatively simple manufacturing demands associated with capillarytube design improves commercial production. Critical dimensions of thedesign are fixed by the high precision of the capillary tubes. Achievingsuitable levels of precision for less critical elements of the design iswithin the capabilities of conventional manufacturing techniques.

Capillary tube printing tip design significantly improves spotmorphology and reproducibility. Printing characteristics of capillarypin printing 6×SSC printing solution were tested using a robot todeposit a CY3 analog to glass over 450 consecutive spots. During courseof a 450 spot deposition pattern, spot fluorescence remains constant.The biggest improvements in using 6×SSC comes in improvements tointra-spot variation. Intra-spot CV improved from approximately 0.75 toabout 0.4. Maintaining consistent concentration and fluid propertiesleads to consistent deposition characteristics. All spots had acoefficient of variations of 5% and 8% for size and deposition volumerespectively.

Inconsistencies in the deposition volume and spot morphology of probespots create variations in probe attachment density. These variationsaffect hybridization parameters which ultimately affect the accuracy ofmicroarray analysis. Capillary tube printing tips were used to print 144spot patterns from a single aliquot of printing solution that containeda unique 465 bp probe DNA. Spots were printed at a relative humidity of70%. After printing, the slides were processed according to recommendedprotocols to prepare for hybridization. All spots were hybridized with asingle aliquot of solution containing two target DNA segments. The firstsegment, complementary to the attached probe, was labeled with Cy-3fluorescent markers. The second segment was not complementary to theattached probe and was labeled with Cy-5 fluorescent markers. Followinghybridization and processing according to recommended protocols, theslides were scanned in a confocal scanner and assessed for levels ofcomplementary and non-complementary hybridization. Although all spotswere printed using the same probe solution and simultaneously hybridizedunder identical conditions, considerable variation in hybridizationlevels and hybridization specificity existed across different spots.Variations in probe attachment density are known to affect levels ofprobe hybridization capacity and specificity. Presumably, variation inprobe deposition resulted in variation in levels of complementary andnon-complementary hybridization. If this is indeed the case, variationsin printing could account for variations of over 45% in microarrayanalysis.

To assist in the development of a capillary tube printing tip of knowncharacteristics, the printing characteristics of a 265 micron (o.d.)glass capillary tube constructed in accordance with the embodiment ofFIGS. 1(a)-1(c) were tested. A robot deposited a CY3 analog to glassover 225 consecutive spots. In this experiment, a borosilicate glasscapillary tube with an inner dimension of 150 um and an outer dimensionof 268 um was cut to a length of approximately 15 mm. The printingcapillary (first tube 24) was fixed with adhesive to the reservoircapillary (second tube 22) with an inner radius of 500 microns. Thecontact surface was polished with 12 microgrit calcite alumina abrasiveto provide a surface that was both flat and hydrophyllic. The pin wasloaded with a 3× solution of SSC buffer that contained dilute Cy-3analog dye (tetramethylrhodamine labeled dextran). The pin was used toprint a 15 by 15 array of spots onto an untreated microscope slide. Theprinted slide was scanned for Cy-3 fluorescence to assess spotmorphology and deposition quantity. The resulting spots printed withvery consistent size and deposition volume. All spots had a coefficientof variations of 5% and 8% for size and deposition volume respectively.

Reproducible design and development techniques can be used to adapt thegeometry of a capillary tube printing tip to a particular microarrayprinting application. Capillary tube tips can be evaluated overdifferent ranges of ambient humidity and duration of pin contact withthe substrate. Variations of spot deposition volume and morphology canbe assessed across consecutive spots printed by a single tip, and acrossspots printed by different tips the same type. Performance of each tipgeometry can be evaluated based on the number of spots that can beprinted from a single loading of printing solution, volume ofdeposition, spot morphology, and consistency of spot deposition andmorphology.

Using video microscopy, deposition volume can be obtained from a shadowprofile of the drop deposited on the slide. Drop volume can becalculated by subtracting the volume of the right cone contained withinthe spherical section outlined by the drop contained on the slidesurface. The images can also be used to compute the contact angle formedby the drop on the slide surface. Preliminary data suggest that thisspherical approximation is quite accurate in describing the shape of thedrop deposited on the slide surface.

In scanner experiments, deposition volumes can be assessed by robotprinting of fluorescently labeled DNA solutions and comparingfluorescence against reference volumes and concentrations. Fluorescenceof printed spots can be assessed by a confocal fluorescence slidescanner. Statistical measurements are performed by automated microarrayanalysis software and include measurements of deposition volume andconsistency of consecutive spots, and uniformity within each spot. Thedeposition volume and morphology of each spot can be assessed for eachpin by printing a 400 spot pattern of solution containing fluorescentlabeled DNA onto glass slides. To facilitate comparison of absolutefluorescence between slides scanned at different sensitivity settings, acalibration curve can be constructed by measuring fluorescence of anarray of Cy-3 concentrations at different scanner settings. All slidescan then be scanned after processing at a laser power of 95% and aphotomultiplier setting of 95% to confirm that the processing protocolsdid not introduce fluorescence to the slides. The total depositionvolume of each spot is then assessed by computing the total fluorescenceas estimated by multiplying the average spot fluorescence times the spotarea and comparing it to total fluorescence of known deposition volumesof reference dye solutions of the same concentration. Measuredparameters include intra-spot fluorescence intensity average andstandard deviation, spot area, inter-spot fluorescence intensity averageand standard deviation for each tip and across multiple tips of the samedesign. The graph below shows the fluorescence of spots deposited by aglass capillary tube printing tip (C.V.=0.08):

Printing tests can be conducted on a microarray printing robot over arange of ambient humidity conditions, using a HEPA filtered humiditycontrolled environment which houses the printing robot. Differentcontact durations of the printing tips with the microarray substrate canbe achieved by adjusting the printing speed of the robot. Observedbehavior is then compared to theoretical predictions to validate designmodels for improved printing pin designs.

Once a minimum contact duration is established for a particularapplication, further testing can focus on the effects of varying ambienthumidity conditions. Reducing the order of experimental variables willsimplify analysis and focus testing on the most significant factorsinfluencing printing behavior.

Transfer of fluid from pins to microarray substrate is controlled by theinterplay of surface tension forces between the pins, substrate, andprinting solution. Surface tension forces within the pin may beestimated by the capillary height equation originally derived by Youngand Laplace.${P_{ambient} - P_{capillary}} = \frac{2\gamma}{r_{capillary}}$Liquid is drawn and maintained inside the capillary lumen by lowpressure achieved by interaction of the fluid with the capillary walls.

Surface tension forces on both the microarray substrate and outersurface of the printing pin may be estimated by analysis of surface freeenergy which gives rise to Young's equation.ΔG=ΔA(γ_(solid-liquid)−γ_(olid=air))+ΔA(γ_(liquid-air) cos(Θ−ΔΘ))A volume of liquid will spread across a surface displacing the surfacefree energy of the substrate with that of the free energy of theliquid-substrate interface until it achieves a state of minimum freeenergy.

The size and volume of a printed spot are the product of the geometry,surface free energy, and liquid surface tension forces which combine toachieve the minimum total free energy of forces between the pin,substrate, and liquid. Detailed analysis of the forces arising fromthese interactions should suggest approaches by which the free energyand geometry of the pin and substrate interface, and the surface tensioncharacteristics of the liquid can be manipulated to achieve the desiredspot characteristics.

Loading pins with printing solution and the solution's subsequentadhesion and spreading on the outer and inner (lumen) pin surface iscontrolled by the surface forces between the pin and liquid, and may beestimated by Young's equation shown above. Pin surfaces with a highsurface free energy promote spreading and adhesion of liquid. Byaltering the pin surface it is possible to change the surface freeenergy and in so doing, change the spreading and adhesion behavior ofliquid in contact with the pin. Several hydrophobic and hydrophilictreatments may be applied to metal and glass pin surfaces to altersurface free energy. The treatments may be used to modulate the boresurface energy from the distal to the proximal ends of the bore.Non-covalent, solvent based treatments include several hand heldhydrophobic markers designed to apply a thin hydrophobic coating.Covalent treatments include silane chemistry in combination with longhydrophobic alkane chains or hydrophilic amine or similarly chargedgroups. Such coatings may even be applied to specific parts of pins topromote specific geometries of spot formation and printing. By strategicplacement of hydrophobic and hydrophilic surface treatments, it shouldbe possible to alter the equilibrium geometry of thepin-substrate-liquid interface, to achieve desired spot characteristics.

As further considerations in the design of a specific capillary tubeprinting tip adapted for a specific microarray printing application, theviscosity of the printing solution and duration of pin contact with thesubstrate will likely affect the time required to establish equilibriumof the printing solution distribution at the pin-substrate contactpoint. It is expected that some minimum time will be required to achieveequilibrium. It expected that printing characteristics will varyconsiderably with combinations of contact duration and viscosity that donot establish equilibrium. Deposition should become more consistent forcombinations of viscosity and contact duration which match or exceed theminimum time to establish equilibrium conditions.

Changing the surface tension of the printing solution is expected toaffect both the volume of deposition and spot morphology. Depositionvolume is likely to be influenced by the equilibrium conditions at thepin-substrate point of contact and liquid surface tension is likely toplay an important role. Spot size is likely to be influenced by thesurface tension of the printing solution on the substrate.

Thus, although there have been described particular embodiments of thepresent invention of a new and useful Capillary Tube Printing Tips forMicroarray Printing, it is not intended that such references beconstrued as limitations upon the scope of this invention except as setforth in the following claims.

1. A printing tip for depositing spots of a liquid material on amicroarray printing substrate comprising: a. a first capillary tubehaving a distal end, a proximal end, and an inner bore, the inner borehaving a distal bore opening at the distal end of the first capillarytube, the inner bore having an axial length and inner diameter adaptedto receive and retain by capillary force an effective deposition volumeof the liquid material; b. the distal end of the first capillary tubedefining an annular contact surface around the distal bore opening, thecontact surface and distal bore opening adapted for drawing the liquidmaterial from the inner bore and depositing a drop of the liquidmaterial on the printing substrate when the contact surface is movedproximate the substrate; and c. gradient means to apply increasedsurface forces to the liquid material within the tip at a distal end ofthe tip compared to a proximal end of the tip.
 2. The printing tip ofclaim 1 wherein the gradient means comprises a first capillary tubehaving an inner bore diameter at the distal end of the tube that issmaller than the inner bore diameter at the proximal end of the tube. 3.The printing tip of claim 1 wherein the gradient means comprises ahydrophilic coating applied to a region of the inner bore proximate thedistal end of the tube.
 4. The printing tip of claim 1 wherein thecontact surface is polished to enhance active wetting of the contactsurface by the liquid material.
 5. The printing tip of claim 1 whereinthe annular contact surface is flat.
 6. The printing tip of claim 1wherein the gradient means comprises a second capillary tube joined toand partially overlapping the distal end of the first capillary tube,the second capillary tube having an inner bore in fluid communicationwith the inner bore of the first capillary tube, and wherein the innerbore of the second capillary tube is functional to act as a reservoirfor the liquid material.
 7. The printing tip of claim 6 wherein thesecond capillary tube is joined to the first capillary tube by anadhesive.
 8. The printing tip of claim 7 wherein the adhesive is heatcured.
 9. The printing tip of claim 7 wherein the adhesive isphoto-cured.
 10. The printing tip of claim 1 wherein the first capillarytube has an outer diameter in the range of 15 to 500 microns.
 11. Theprinting tip of claim 10 wherein the inner bore of the first capillarytube has an inner diameter in the range of 5 to 250 microns.
 12. Theprinting tip of claim 10 wherein the inner bore of the first capillarytube has an axial length in the range of 500 to 1500 microns.
 13. A tipfor depositing a liquid material on a microarray printing substratecomprising: a. a reservoir section; b. a contact section having aproximal end and a distal end, the proximal end of the contact sectionjoined to the reservoir section, the contact section further comprisingan inner bore extending upward from a bore opening at the distal and ofthe contact section, the bore in fluid communication with the reservoirsection, the distal end of the contact section further defining acontact surface adjacent the bore opening; c. the reservoir section isadapted to receive and retain by capillary force an effective depositionvolume of the liquid material; and d. the contact surface and boreopening are adapted for depositing a drop of the liquid material whenthe contact surface is moved proximate the printing substrate.
 14. Theprinting tip of claim 13 wherein the reservoir section comprises aproximal region of the inner bore having an inner diameter the is largerthan the inner diameter of the inner bore proximate the contact surface.15. The printing tip of claim 13 wherein the contact surface is flat.16. A contact printing tip for printing a spot of a liquid material on amicroarray printing substrate comprising: a. first and second concentriccapillary tubes, the second capillary tube having an inner bore with anaxial length, and an inner diameter that is larger than an outerdiameter of the first capillary tube so that the second capillary tubepartially overlaps a proximal end of the first capillary tube; b. thefirst capillary tube having an inner bore in fluid communication withthe inner bore of the second capillary tube; c. the first capillary tubefurther comprising a contact surface at a distal end of the firstcapillary tube, the contact surface surrounding an opening from theinner bore of the first capillary tube; d. the inner bore of the secondcapillary tube having an axial length, an inner diameter, and inner boresurface adapted to receive and retain an amount of the liquid material;and e. the inner bore of the first capillary tube having an axiallength, an inner diameter, and an inner bore surface adapted for drawingthe liquid material retained in inner bore of the second capillary tubeby capillary action and depositing a drop of the liquid material on theprinting substrate when the contact surface is moved proximate thesubstrate.
 17. The printing tip of claim 16 wherein the inner bore ofthe first capillary tube has an inner diameter in the range of 5 to 250microns.
 18. The printing tip of claim 17 wherein the inner bore of thesecond capillary tube has an inner diameter in the range of 25 to 4000microns.
 19. The printing tip of claim 18 wherein the inner bore of thefirst capillary tube has an axial length in the range of 500 to 1500microns.
 20. The printing tip of claim 19 wherein the inner bore of thesecond capillary tube has an axial length in the range of 500 to 4000microns.
 21. The printing tip of claim 16 wherein the first capillarytube comprises stainless steel.